Recently, a next-generation sequencing (NGS)-based method has been used for the successful detection of circulating tumor DNA (ctDNA) in various cancer types. Thus, the use of NGS on liquid biopsies will improve cancer diagnosis and prognosis. However, the low-allelic fraction of ctDNA poses a challenge for the sensitive and specific detection of tumor variants in cell-free DNA (cfDNA). To distinguish true variants from false positives, the characteristics of errors that occur during sample preparation and sequencing need to be elucidated. We generated capture-based targeted deep sequencing data from plasma cfDNA and peripheral blood leucocyte (PBL) gDNA to profile background errors. To reveal cfDNA-associated DNA lesions, background error profiles from two sample types were compared in each nucleotide substitution class. In this study, we determined the prevalence of single nucleotide substitutions in cfDNA sequencing data to identify DNA damage preferentially associated with cfDNA. On comparing sequencing errors between cfDNA and cellular genomic DNA (gDNA), we observed that the total substitution error rates in cfDNA were significantly higher than those in gDNA. When the substitution errors were divided into 12 substitution error classes, C:G>T:A substitution errors constituted the largest difference between cfDNA and gDNA samples. When the substitution error rates were estimated based on the location of DNA-fragment substitutions, the differences in error rates of most substitution classes between cfDNA and gDNA samples were observed only at the ends of the DNA fragments. In contrast, C:G>T:A substitution errors in the cfDNA samples were not particularly associated with DNA-fragment ends. All observations were verified in an independent dataset. Our data suggested that cytosine deamination increased in cfDNA compared to that in cellular gDNA. Such an observation might be due to the attenuation of DNA damage repair before the release of cfDNA and/or the accumulation of cytosine deamination after it. These findings can contribute to a better understanding of cfDNA-associated DNA damage, which will enable the accurate analysis of somatic variants present in cfDNA at an extremely low frequency.

中文翻译：

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通过靶向深度测序表征与游离 DNA 相关的 DNA 损伤


最近，基于下一代测序 (NGS) 的方法已被用于成功检测各种癌症类型中的循环肿瘤 DNA (ctDNA)。因此，在液体活检中使用NGS将改善癌症诊断和预后。然而，ctDNA 的低等位基因部分对无细胞 DNA (cfDNA) 中肿瘤变异的灵敏和特异性检测提出了挑战。为了区分真正的变异和假阳性，需要阐明样品制备和测序过程中发生的错误的特征。我们从血浆 cfDNA 和外周血白细胞 (PBL) gDNA 生成基于捕获的靶向深度测序数据，以分析背景错误。为了揭示 cfDNA 相关的 DNA 损伤，在每个核苷酸替换类别中比较了两种样本类型的背景错误概况。在这项研究中，我们确定了 cfDNA 测序数据中单核苷酸替换的发生率，以识别优先与 cfDNA 相关的 DNA 损伤。在比较 cfDNA 和细胞基因组 DNA (gDNA) 之间的测序错误时，我们观察到 cfDNA 中的总替换错误率显着高于 gDNA。当替换错误分为12个替换错误类别时，C:G>T:A替换错误构成了cfDNA和gDNA样本之间的最大差异。当根据 DNA 片段替换的位置估计替换错误率时，仅在 DNA 片段的末端观察到 cfDNA 和 gDNA 样本之间大多数替换类别的错误率差异。相比之下，cfDNA 样品中的 C:G>T:A 替换错误与 DNA 片段末端没有特别相关。所有观察结果均在独立数据集中得到验证。 我们的数据表明，与细胞 gDNA 相比，cfDNA 中的胞嘧啶脱氨基作用有所增加。这样的观察结果可能是由于 cfDNA 释放之前 DNA 损伤修复的减弱和/或之后胞嘧啶脱氨作用的积累。这些发现有助于更好地了解 cfDNA 相关的 DNA 损伤，从而能够以极低的频率准确分析 cfDNA 中存在的体细胞变异。